Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection

ABSTRACT

The present invention provides for a method to circumvent the problem of using antibiotic resistant selectable markers. In particular, target plants are transformed using a plastid vector which contains heterologous DNA sequences coding for a phytotoxin detoxifying enzyme or protein. The selection process involves converting a antibiotic-free phytotoxic agent by the expressed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides for various methods to use antibiotic-free selection in chloroplast transformation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application Ser. No.09/807,722; filed Apr. 18, 2001 (now abandoned); which application was anational stage application of international application PCT/US01/06275;filed Feb. 28, 2001; which claims the benefit of provisional patentapplications Ser. No. 60/259,154; filed Dec. 28, 2000; Ser. No.60/257,406; filed Dec. 22, 2000; Ser. No. 60/209,762; filed Jun. 6,2000; and Ser. No. 60/186,308; filed Mar. 2, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work of this invention is supported in part by the USDA-NRICGPgrants 95-82770, 97-35504 and 98-0185 to Henry Daniell.

FIELD OF THE INVENTION

This application pertains to the field of genetic engineering of plantplastid genomes, particularly chloroplasts, and to methods of andengineered plants without the use of antibiotics.

This application relates in particular to a method of selectinggenetically engineered or transformed plants without the use ofantibiotics as a selectable marker. The application also relates to amethod of transforming plants to confer drought tolerance and to thetransformed plants which are drought tolerant.

DESCRIPTION OF THE RELATED ART

Publications

Various methods of selection of plants that employ antibiotic-freeselectable marker, or non-antibiotic selectable markers, have beendescribed in the past.

Briggs, in U.S. Pat. No. 5,589,611 (Dec. 31, 1996) entitled “Diseaseresistance gene from maize and its use for disease resistance as aselectable marker and as a gene identification probe,” proposed a methodof identifying transformed plants which is disease resistant. A genethat controls resistance to both a fungus and a fungal disease toxin isproposed as a selectable marker to identify transformed plants,particularly in maize. An expression cassette containing the DNAsequence of a disease resistance gene, namely the Hm1 gene in maize, isinserted into the nucleic genome of the plant cells. The transformedplants will be capable of producing HC-toxin reductase. By culturing thecells in growth medium containing the corresponding toxin produced bythe pathogen, namely Cocholiobolus carbonum Nelson race 1, the lethalselection of transformed plants will result.

Ursin, in U.S. Pat. No. 5,633,153 (May 27, 1997) entitled “Aldehydedehydrogenase selectable markers for plant transformation,” proposed amethod of using an aldehyde dehydrogenase as a selectable marker fornuclear transgenic plant cells. A DNA construct coded for an aldehydedehydrogenase through eukaryotic promoters used for nucleartransformation and culturing such transformed cells in growth mediacontaining the corresponding phytotoxic aldehyde, the transformed plantsdemonstrate resistance to the phytotoxic aldehyde.

Song, in U.S. Pat. No. 5,965,727 (Oct. 12, 1999), entitled “Forselectable markers and promoters for plant tissue culturetransformation,” proposed transforming nuclear genome of plant cellswith an expression cassette which contains DNA sequences coded for boththe ASA2 promoter sequence of Nicotiana tabacum, or fragments thereof,that are capable of directing tissue culture specific expression. TheASA2 gene which is substantially resistant to inhibition by free L-Trpor an amino acid analog of Trp. When such cells are cultured in a mediumcontaining an amount of an amino acid analog of Trp, successfullytransformed plant cells survive.

Several patents have also discussed the conferring of osmoprotection toplants through plant transformation. Adams, in U.S. Pat. No. 5,780,709(Jul. 14, 1998) entitled “Transgenic maize with increased mannitolcontent”, proposed a method of conferring resistance to water or saltstress or altering the osmoprotectant content of a monocot plant bynucleic transformation. Transformation is accomplished via a vectorcontaining an expression cassette comprised of a preselected DNA segmentcombined with a eukaryotic promoter functional in plant nucleus. Thus,the preselected DNA segment that was used to transform the monocotplants was the mt1D gene which encodes for the enzyme that catalyzes thesynthesis of mannitol. Adams focused on the osmoprotective properties ofsugar alcohols, specifically mannitol.

Wu, in U.S. Pat. No. 5,981,842 (Nov. 9, 1999), proposed thatosmoprotection can be conferred upon cereal plants by transformingcereal plant cells or protoplasts with a promoter and a nucleic acidencoding a group 3 late embryogenesis protein (LEA protein) such as theHVA1 gene from barley. The transformed cereal plant accumulates HVA1protein in both leaves and roots. The transformed plants showed anincrease tolerance to drought and salt stress which correlated with thelevel of the HVA1 protein accumulated in the transformed plants.

All publications and patent applications are herein incorporated byreference.

BACKGROUND OF THE INVENTION

Disadvantages of the antibiotic selectable marker system. Mosttransformation techniques co-introduce a gene that confers antibioticresistance, along with the gene of interest to impart a desired trait.Regenerating transformed cells in antibiotic containing growth mediapermits selection of only those cells that have incorporated the foreigngenes as the gene of interest. Once transgenic plants are regenerated,antibiotic resistance genes serve no useful purpose but they continue toproduce their gene products. One of the primary concerns of geneticallymodified (GM) crops is the presence of clinically important antibioticresistance gene products in transgenic plants that could inactivate oraldoses of the antibiotic (reviewed by Puchta 2000; Daniell 1999A).Another concern is that the antibiotic resistant genes could betransferred to pathogenic microbes in the gastrointestinal tract or soilrendering them resistant to treatment with such antibiotics. Antibioticresistant bacteria are one of the major challenges of modern medicine.In Germany, GM crops containing antibiotic resistant genes have beenbanned from release (Peerenboom 2000).

Plastid genetic engineering as an alternative to nuclear geneticengineering. Plastid genetic engineering, particularly chloroplastgenetic engineering, is emerging as an alternative new technology toovercome some of the environmental concerns of nuclear geneticengineering (reviewed by Bogorad, 2000). One common environmentalconcern is the escape of foreign gene through pollen or seed dispersalfrom transgenic crop plants to their weedy relatives creating superweeds or causing genetic pollution among other crops (Daniell 1999B).Keeler et al. (1996) have summarized valuable data on the weedy wildrelatives of sixty important crop plants and potential hybridizationbetween crops and wild relatives. Among sixty crops, only eleven do nothave congeners and the rest of the crops have wild relatives somewherein the world. In addition, genetic pollution among crops has resulted inseveral lawsuits and shrunk the European market of Canadian organicfarmers (Hoyle 1999). Several major food corporations have requiredsegregation of native crops from those “polluted” with transgenes. Twolegislations have been submitted in the U.S. to protect organic farmerswhose crops inadvertently contain transgenes via pollen drift (Fox2000). Maternal inheritance of foreign genes through chloroplast geneticengineering is highly desirable in such instances where there ispotential for out-cross among crops or between crops and weeds (Daniellet al. 1998; Scott and Wilkinson 1999; Daniell 1999C).

Yet another concern in the use of nuclear transgenic crops expressingthe Bacillus thuringiensis (Bt) toxins is the sub-optimal production oftoxins resulting in increased risk of pests developing Bt resistance.Plant-specific recommendations to reduce Bt resistance developmentinclude increasing Bt expression levels (high dose strategy), expressingmultiple toxins (gene pyramiding), or expressing the protein only intissues highly sensitive to damage (tissue specific expression). Allthree approaches are attainable through chloroplast transformation(Daniell 1999C). For example, hyperexpression of several thousand copiesof a novel B.t. gene via chloroplast genetic engineering, resulted in100% mortality of insects that are up to 40.000-fold resistant to otherB.t. proteins (Kota et al. 1999). Another hotly debated environmentalconcern expressed recently is the toxicity of transgenic pollen tonon-target insects, such as the Monarch butterflies (Losey et al. 1999;Hodgson 1999). Although pollen from a few plants shown to exhibitmaternal plastid inheritance contains metabolically active plastids, theplastid DNA itself is lost during the process of pollen maturation andhence is not transmitted to the next generation (reviewed in Heifetz,2000, Bock and Hagmann, 2000). Lack of insecticidal protein intransgenic pollen engineered via the chloroplast genome with the cry2Agene has been demonstrated recently, even though chloroplast in leavescontained as much as 47% CRY protein of the total soluble protein (DeCosa et al. 2000).

The need for alternatives to the antibiotic selectable marker system.Despite these advantages, one major disadvantage with chloroplast:genetic engineering in higher plants may be the utilization of theantibiotic resistance genes as the selectable marker to conferstreptomycin/spectinomycin resistance. Initially, selection forchloroplast transformation utilized a cloned mutant 16S rRNA gene thatdoes not bind the antibiotic and this conferred spectinomycin resistance(Svab et al. 1990). Subsequently, the aadA gene product that inactivatesthe antibiotic by transferring the adenyl moiety of ATP tospectinomycin/streptomycin was used (Svab and Maliga 1993). Theseantibiotics are commonly used to control bacterial infection in humansand animals. The probability of gene transfer from plants to bacterialiving in the gastrointestinal tract or soil may be enhanced by thecompatible protein synthetic machinery between chloroplasts andbacteria, in addition to presence of thousands of copies of theantibiotic resistance genes per cell. Also, most antibiotic resistancegenes used in genetic engineering originate from bacteria.

Because of the presence of thousands of antibiotic resistant genes ineach cell of chloroplast transgenic plants and the use of the mostcommonly used antibiotics in the selection process, it is important todevelop a chloroplast genetic engineering approach without the use ofantibiotics.

Non-obviousness of antibiotic free selection. Despite several advantagesof plastid transformation, one major disadvantage with chloroplastgenetic engineering in higher plants is the utilization of theantibiotic resistance genes as the selectable marker. Initially,selection for chloroplast transformation utilized a cloned mutant 16SrRNA gene that did not bind the antibiotic and this conferredspectinomycin resistance. Subsequently, the aadA gene was used as aselectable marker. Aminoglycoside 3′-adenylyltransferase inactivates theantibiotic by transferring the adenyl moiety of ATP tospectinomycin/streptomycin. Unfortunately, bacterial infections inhumans and animals are also controlled by using these antibiotics. Theprobability of gene transfer from plants to bacteria living in the soilor gastrointestinal tract may be enhanced by the compatible proteinsynthetic machinery between chloroplasts and bacteria, in addition topresence of thousands of copies of the antibiotic resistance genes percell. Also, most antibiotic resistance genes used in genetic engineeringoriginate from bacteria.

Prior to this invention, there was no report of modifying the plastidgenome without the use of antibiotic selection. Daniell et al. (2001)reported the first genetic engineering of the higher plant chloroplastgenome without the use of antibiotic selection. The betaine aldehydedehydrogenase (BADH) gene from spinach was used as a selectable marker.The selection process involves conversion of toxic betaine aldehyde (BA)by the BADH enzyme to nontoxic glycine betaine, which also serves as anosmoprotectant. While it was known earlier that BADH was a plant enzyme,it could not be conclusively demonstrated that this was a chloroplastenzyme because it lacked the typical transit peptide found in allchloroplast proteins imported from the cytosol.

The absence of a typical transit peptide raised several questions aboutproper cleavage of BADH enzyme in the stroma within plastids to be fullyfunctional. It was not known whether the BADH enzyme would becatalytically active without proper cleavage within plastids.

The nuclear BADH cDNA with high GC content was never anticipated toexpress well in the AT rich prokaryotic plastid compartment because thecodon usage is very different between the prokaryotic chloroplastcompartment and the eukaryotic nuclear compartment. Therefore, it wasnot obvious to express a nuclear gene in the plastid compartment.

When the chloroplast transformation system was developed, it washypothesized that the transformation process is possible only undernon-lethal selection. Accumulation of betaine aldehyde is toxic andlethal to plant cells. Therefore, it was not clear whether non-lethalselection was required for chloroplast transformation. This inventionhas confirmed that the only requirement was that the selection processshould be specific to plastids, particularly chloroplasts.

Rapid regeneration of chloroplast transgenic plants obtained under BAselection was never anticipated or suggested in any prior art.Chloroplast transformation efficiency was 25 fold higher in BA selectionthan spectinomycin and this was never anticipated in any previousinvestigations. Higher efficiency of betaine aldehyde selection comparedto spectinomycin should facilitate chloroplast transformation of manyeconomically important crops, including cereals that are naturallyresistant to spectinomycin, in addition to conferring salt/droughttolerance.

Use of genes that are naturally present in spinach for selection, inaddition to gene containment, should ease public concerns regarding GMcrops.

SUMMARY OF THE INVENTION

The invention provides for a method to circumvent the problem of geneticpollution through plastid transformation and the use antibiotic-freeselectable markers. Antibiotic-free phytotoxic agents and theircorresponding detoxifying enzymes or proteins are used as a system ofselection. In particular, the betaine aldehyde dehydrogenase (BADH) genefrom spinach has been used as a selectable marker. This enzyme ispresent only in chloroplasts of a few plant species adapted to dry andsaline environments. The selection process involves conversion of toxicbetaine aldehyde (BA) by the chloroplast BADH enzyme to nontoxic glycinebetaine (GB), which also serves as an osmoprotectant.

The preferred embodiment of this invention provides a method ofselecting plant transformants using a plastid vector that includes apromoter targeted to the plastid, a DNA sequence encoding a gene ofinterest, another DNA sequence encoding a selectable marker such as analdehyde dehydrogenase, and a terminator sequence. The transformedplants are selected by allowing transformed plants to grow in mediumwith the effective amount of a phytotoxin which is detoxified by theencoded aldehyde dehydrogenase. Lethal selection of the plantstransformants will result.

It is another embodiment of this invention, the vector is targeted toplant chloroplasts. This embodiment can be carried out using both theuniversal chloroplast vector and a vector which is universal.Preferably, the vector includes a ribosome binding site and a 5′untranslated region (5′ UTR. A promoter functional in green or non-greenplastids is to be used in conjunction with the 5′UTR

The invention provides the application of a wide variety of plantsspecies and plant parts, including flowers, fruits, cereals, and allmajor crop plants.

The invention also provides for the plants transformants engineered andselected a antibiotic-free selectable marker with preferably a targetheterologous DNA sequence.

The invention also provides for a method of conferring drought toleranceto plants with a antibiotic-free selectable marker. The plants or plantcells are transformed through the chloroplast by a vector containing apromoter targeted to the chloroplast, a DNA sequence encoding betainealdehyde dehydrogenase, DNA sequences encoding at least one gene ofinterest, and a terminator sequence. The transformed plants are selectedby allowing transformed plants to grow in medium with the effectiveamount of a phytotoxin which is detoxified by the encoded aldehydedehydrogenase. Lethal selection of the plants transformants will result.The plants so transformed will be capable of glycine betaine productionthat leads to enhanced drought tolerance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chloroplast universal vector pLD BADH. Primer 3P landson the native chloroplast genome (in the 5′ end region of 16-S r DNAgene). 3M lands on the aadA gene generating a 1.6 kb fragment.Restriction enzyme cut site are located on the map.

FIG. 2 shows BADH enzyme activity in E. coli. Cells harvested fromovernight grown cultures were resuspended in a minimal volume of theassay buffer. Sonicated cell homogenate was desalted in G-25 columns and50 μg total protein was used fr each assay. NAD+ dependent BADH enzymewas analyzed for the formation of NADH by increase in the absorbency at340 nm.

FIG. 3 shows a comparison of betaine aldehyde and spectinomycinselection. A. N. tabacum Petit Havana control in RMOP medium containingspectinomycin after 45 days. B. Bombarded leaf discs selected onspectinomycin in RMOP medium after 45 days. C. Spectinomycin resistantclones cultured again (sound round) to obtain homoplasmy. D. PetitHavana control in RMOP medium containing betaine aldehyde after 12 daysof culture. E. Bombarded leaf discs selected on betaine aldehyde in RMOPmedium after 12 days of culture; arrow indicates unbombarded leaf discas control. Note that 23 shoots are formed on a disc selected on betainaldehyde against 1-2 shoots per disc on spectinomycin. F. Betainealdehyde resistant clones cultureed again (second round) to obtainhomoplasmy. G. Selection on 10 mM betaine aldehyde of untransformed (1)and transgenic (2-4) leaf discs. Note shoots from transgenic leaf discsand death of untransformed leaf disc.

FIG. 4 shows the PCR analysis of DNA extracted from transformed plantsrun on a 0.8% agarose gel. Lane M 1 kb ladder, lane 1, untransformedPetit Havana control, lane 17 is positive control and lanes 2 through 16are transgenic clones. Except lanes 10, 13, 15 and 16 all other lanesshow the integration of aadA gene into the chloroplast genome.

FIG. 5 shows the Southern analysis of transgenic plants. A: Probe P1 wasused to confirm chloroplast integration of foreign genes. The 0.81 kbfragment was cut with BamHl and Bglll contains the flanking sequenceused for homologous recombination. Untransformed control plants shuoldgenerate 4.47 kb fragment and transformed plants should generate a 7.29kb fragment. B: Lanes 1, untransformed Petit Havana; Lanes 7 pLD-BADHplasmid DNA or purified DNA or purified 1.0 kb Eco R1 BADH genefragment. Lanes 2 through 6 of transgenic plants. Probe (P2) was used tconfirm the integration of BADH gene.

FIG. 6 shows BADH enzyme activity in different ages of leaves oftransgenic tobacco plant. Proteins were extracted from 1-2 g leaves.Extracts were centrifuged at 10,000×G for 10 minutes and the resultingsupernatant was desalted in small G-25 columns, and tested for assay (50μg protein per assay). NAD+ dependent BADH enzyme was analyzed for theformation of NADH. Y, D, M and O represent young, developing, mature andold leaves, respectively.

FIG. 7 shows the phenotypes of control (A) and chloroplast transgenicplants (B).

FIG. 8 shows the germination of control untransformed (a) andchloroplast transgenic (b) seeds on MS medium containing 500 μg/mlspectinomycin.

FIGS. 9A and B show the vectors for BADH selection in other plants.

Table 1 shows the comparison of spectinomycin and betaine aldehyde asthe selectable marker for the first round of selection.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a nucleotide sequence of primer 3M.

SEQ ID NO: 2: is a nucleotide sequence of primer 3P.

DETAILED DESCRIPTION OF THE INVENTION

The invention discloses a novel way of selecting transformed plants,wherein the plant's plastid genome is transformed via a vector targetedto the plastid, and the selectable markers used for such transformationis a antibiotic-free marker. The invention further consists of theplants transformed and selected using the present method. The inventionalso discloses a method to confer osmoprotection to plants throughchloroplast transformation.

The present invention is applicable to all plastids of plants. Theseinclude chromoplasts which are present in the fruits, vegetables andflowers; amyloplasts which are present in tubers like the potato;proplastids in roots; leucoplasts and etioplasts, both of which arepresent in non-green parts of plants.

The Vectors. This invention contemplates the use of vectors capable ofplastid transformation, particularly of chloroplast transformation. Suchvectors would include chloroplast expression vectors such as pUC,pBR322, pBlueScript, pGEM, and all others identified by Daniell in U.S.Pat. No. 5,693,507 and U.S. Pat. No. 5,932,479. Included are alsovectors whose flanking sequence is located outside the inverted repeatof the chloroplast genome. These publications and patents are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

A preferred embodiment of this invention utilizes a universalintegration and expression vector competent for stably transforming thechloroplast genome of different plant species (Universal Vector). Auniversal vector is described in WO 99/10513 which was published on Mar.4, 1999, which is herein incorporated in its entity.

The vector pLD-BADH was constructed by generating a PCR product usingspinach cDNA clone as the template. The 5′ primer also included thechloroplast optimal ribosome binding site (GGAGG). PCR product wassubcloned into the EcoR1 site of pLD-CtV, resulting in pLD-BADH. BADH isone of the few proteins targeted to the chloroplast that lacks adefinite transit peptide (Rathinasabapathi et al 1994). Authors suggestthat information for transport may be contained within the matureprotein. Even if a transit peptide was present, it should be cleaved inthe stroma by the stromal processing peptidase (Keegstra and Cline,1999). Furthermore, nuclear encoded cytosolic proteins with transitpeptides have been successfully expressed within chloroplasts and foundto be fully functional (Daniell et al. 1998). Therefore there was noneed to delete any transit peptide.

The universal vector, pLD-BADH, as shown in FIG. 1, integrates the aadAand BADH genes into the 16S-23S-spacer region of the chloroplast genome.Expression cassettes of the chloroplast integration vector contain thechimeric aadA gene and the BADH gene driven by the constitutive 16S rRNApromoter and regulated by the 3′ untranslated region of the plastid psbAgene. The chimeric aadA gene encoding aminoglycoside 3′adenyltransferaseconfers spectinomycin resistance in chloroplasts enabling selection ofthe transformants on spectinomycin dihydrochloride. On the other hand,BADH converts the toxic betaine aldehyde in cells to glycine betaine.When present, this pathway is compartmentalized within chloroplasts(Nuccio, et al. 1999). To facilitate translation of the dicistronicmRNA, independent Shine-Dalgarno (SD) sequences were provided to theaadA and BADH genes upstream of the initiation codons. In order toaccurately compare transformation efficiency of both selectable markersunder identical bombardment and transformation conditions, aadA and BADHgenes were inserted into the same vector, at the same site. Bombardedleaves were treated in identical manner except the addition of selectionreagent.

Other plant specific vectors can be used to transform the plastids,particularly chloroplast, of various crops for betaine aldehydeselection. Some examples of these include the pLD-Alfa-BADH is fortransforming the chloroplast genome of Alfalfa using betaine aldehydeselection; the pLD-Gm-utr-BADH is for transforming the chloroplastgenome of Soybean (Glycine max) with betaine aldehyde; this contains thepsbA promoter and untranslated region (UTR) for enhanced expression; thepLD-St-BADH is for transforming the chloroplast genome of potato(Solanum tuberosum) using betaine aldehyde selection; pLD-St-utr-BADH isfor transforming the chloroplast genome of potato (Solanum tuberosum)with betaine aldehyde; this contains the psbA promoter and untranslatedregion (UTR) for enhanced expression; and the pLD-Tom-BADH is fortransforming the chloroplast genome of tomato using betaine aldehydeselection.

Promoters. For transcription and translation of the DNA sequenceencoding the gene of interest, the entire promoter region from a genecapable of expression in the plastid generally is used. The promoterregion may include promoters obtained from green and non-greenchloroplast genes that are operative upon the chloroplast, such as thepsbA gene from spinach or pea, the rbcL, atpB promoter region frommaize, the accD promoter and 16S rRNA promoter. Competent promoters arealso described in U.S. Pat. No. 5,693,507, and the other literaturesources contained therein. These publications and patents are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. Selectable markers. The preferred embodimentof this invention teaches the use of the spinach BADH gene as aselectable marker; wherein a plant is transformed via the chloroplastwith the spinach BADH gene along with another nucleotide sequenceencoding a desirable trait. The BADH gene product—betaine aldehydedehydrogenase—will oxidize the betaine aldehyde in the growth mediumallowing for the lethal selection of transformed plants.

Other forms of Antibiotic-Free Selection. Enzymes and proteins thatfunction in plastids can be used as antibiotic-free phytotoxic agents.In case of amino acid biosynthesis, the synthesis is regulated by thesubstrate. When adequate amino acid is made, it binds to one of theenzymes in the pathway to block further synthesis (feed backinhibition). Mutant genes are available for many enzymes that areinsensitive to such feed back inhibition. Such enzymes are expressed inthe chloroplast by engineering feed back insensitive mutant genes viathe chloroplast genome. Putative transgenic shoots are regenerated in agrowth medium lacking specific amino acids. True transgenic plants willbe regenerated in the growth medium. Thus, antibiotic free selection isaccomplished.

Pigment biosynthesis can also be used in antibiotic free selection inplastids. While ancient plants (including pines) have the ability tosynthesize chloroplhyll in the dark, flowering plants lost thiscapacity. This is because of the last step in chlorophyll biosynthesisis controlled by the enzyme protochlorophyllide reductase. This enzymecan function in the dark in primitive land plants and certain algae butis light dependent in higher plants. That is why ornamental plants keptinside the house requires light to synthesize chlorophyll. It is knownthat the chloroplast gene (chlB) for protochlorophyllide reductase inthe green alga Chlamydomonas is required for light independentprotochlorophyllide reductase activity (Plant Cell 5: 1817-1829).Therefore, chlB gene from the Chlamydomonas chloroplast is introducedinto the chloroplast genome of higher plants and transgenic green shootsappearing in the dark is selected. Thus, pigment biosynthesis genes areused as antibiotic free selectable markers.

Another possibility is herbicide selection. Several methods can be usedto genetically engineer herbicide resistance via the chloroplast genome.The target enzyme or protein is overproduced with 10,000 copies offoreign genes per transformed cell. This results in binding of allherbicide molecules thereby facilitating regeneration of transgenicshoots. Another approach is the use of modified enzyme or proteins(mutant) that does not bind the herbicide. The third approach is to useenzymes or proteins to breakdown the herbicide.

Drought tolerance likewise can be used as a selectable marker.Expression of the BADH enzyme or trehalose phosphate synthase via thechloroplast genome enables cells to tolerate drought. Drought conditionsare created in culture plates by the addition of polyethylene glycol tothe growth medium (3-6%). Only cells that express BADH or TPS arecapable of drought tolerance and grows in the presence of polyethyleneglycol. Thus, antibiotic free chloroplast transgenic plants areobtained.

Other Aldehyde Dehydrogenases. Other genes that code for an aldehydedehydrogenase capable of detoxifying other phytotoxic aldehydes can beused in this novel selection system. These include, and are not limitedto, genes that encode acetaldehyde dehydrogenase, formaldehydedehydrogenase, proprionaldehyde dehydrogenase, and butyraldehydedehydrogenase.

Plastid Transformation

The transformation of this invention maybe accomplished by any methodsof transformation known in the art. Such methods include, but are notlimited to PEG treatment, Agrobacterium treatment, and microinjection.Methods of transformation are described by Daniell et. al., “New Toolsfor Chloroplast Genetic Engineering,” Nat. Biotechnology, 17:855-857(1999). This publication is hereby incorporated by reference in itsentirety. In the preferred embodiment, the method for transformation isby bombardment.

The BADH gene expression was tested in E. coli cell extracts by enzymeassays before proceeding with bombardment. The universal vector pLD-BADHwas transformed into the E. coli strain XL-1 Blue and grown in TerrificBroth (Guda et al. 2000) in the presence of ampicillin (100 μg/ml) at37° C. for 24 hours. In E. coli, the level of expression by thechloroplast Prrn promoter is equivalent to that of the highly efficientT7 promoter and both systems have highly compatible protein syntheticmachinery (Brixey et al. 1997). Therefore, BADH enzyme activity wastested in untransformed cells and cells transformed with pLD-BADH, ahigh copy number plasmid (FIG. 2). Crude sonic extracts isolated fromtransformed cells showed 3-5 fold more BADH activity than theuntransformed control, confirming that the expression cassette is fullyfunctional. This result also suggests that codon preference of thenuclear BADH gene is compatible with expression in the prokaryoticchloroplast compartment.

Tobacco (Nicotiana tabacum var. Petit Havana) was grown aseptically bygermination of seeds in MSO medium. This medium contains MS salts (4.3g/liter), B5 vitamin mixture (myoinositol, 100 mg/liter; thiamine-HCl,10 mg/liter; nicotinic acid, 1 mg/liter; pyridoxine-HCI, 1 mg/liter),sucrose (30 g/liter) and phytagar (6 g/liter) at pH 5.8. Fully expanded,dark green leaves of about two month old plants were used forbombardment.

Leaves were placed abaxial side up on Whatman No. 1 filter papers layingon the RMOP medium (Daniell 1993) in standard petri plates (100×15 mm)for bombardment. Microprojectiles were coated with plasmid DNA(pLD-BADH) and bombardments were carried out with the biolistic devicePDS 1000/He (Bio-Rad) as described by Daniell (1997). Followingbombardment, petri plates were sealed with parafilm and incubated at 24°C. under 16 hour photoperiod. Two days after bombardment, leaves werechopped into small pieces of ˜5 mm² in size and placed on the selectionmedium (RMOP containing 500 μg/ml of spectinomycin dihydrochloride or5-10 mM betaine aldehyde) with abaxial side touching the medium in deep(100×25 mm) petri plates. The regenerated resistant shoots were choppedinto small pieces (˜2 mm²) and subcloned into fresh deep petri platescontaining the same selection medium. Resistant shoots from the secondculture cycle were transferred to the rooting medium (MSO mediumsupplemented with IBA, 1 mg/liter containing appropriate selectablemarker). Rooted plants were transferred to soil and grown at 26° C.under 16 hour photoperiod.

Selection and Heightened, Rapid Regeneration of Homoplasmic TransgenicPlants.

The entire process of regeneration, starting from bombardment untiltransfer to soil, takes about 3-6 months for spectinomycin selection and2-3 months for betaine aldehyde selection. FIG. 3 and Table 1 showdifferences between the two selection processes. Under spectinomycinselection, leaf discs continued to grow but pigments were bleached;resistant clones formed green shoots in about 45 days (FIG. 3B). On theother hand, under betaine aldehyde selection, growth of the leaf discswas completely inhibited and photosynthetic pigments were degraded (FIG.3G-1), resistant clones formed green shoots within 12 days (FIG. 3E).Leaf disks in FIG. 3 under betaine aldehyde selection appear partiallygreen because they were photographed 12 days after the initiation of theselection process whereas the disc photographed on spectinomycin were 45days after initiation of the selection process. In spite of the shortperiod of selection one leaf disk was almost bleached (FIG. 3D) and allof them were killed after 30 days. Under 10 mM betaine aldehydeselection, control untransformed samples were killed (turned black,3G-1) whereas transgenic leaves produced new shoots (FIG. 3G, 2-4).

When the leaf discs were selected for spectinomycin resistance, only 15%of the discs responded and an average of one resistant shoot per platewas observed after 45 days. From each callus, all resistant shoots areconsidered to represent an individual clone. Under betaine aldehydeselection 80% of the discs responded and an average of 25 resistantshoots per plate was observed. Responding leaf disks formed one or tworesistant shoots under spectinomycin selection whereas under betainealdehyde selection, as many as 23 shoots were observed from a singleleaf disk. Overall, 10 resistant shoots were regenerated from tenbombardments under spectinomycin selection while more than 150 shootswere recovered from six bombardments under betaine aldehyde selection.Therefore, the efficiency of transformation is 25 fold higher in betainealdehyde selection than spectinomycin selection. Additionally, thelatter procedure results in rapid regeneration.

Lethal Selection. The prior art suggests that chloroplast transformationsystem is possible only under non-lethal selection (Svab and Maliga1993). This invention distinctly shows that this is not the case.Non-lethal selection was defined in the chloroplast transformationliterature as lack of suppression of growth on the selection medium andthat this was an absolute requirement for plastid transformation (Stauband Maliga 1993). It is known that accumulation of betaine aldehyde istoxic and lethal to plant cells (Rathinasabapathi et. al. 1994). Thisinvention confirm earlier observations that betaine aldehyde is toxic toplant cells and inhibits growth. Therefore, this invention teaches thatnon-lethal selection is not a requirement for plastid transformation.The only requirement is that the selection process should be specific toplastids.

Confirmation of Chloroplast Integration, Homoplasmy and Copy Number.

Integration of a foreign gene into the chloroplast genome was confirmedby PCR screening of chloroplast transformants (FIG. 4). Primers weredesigned to eliminate mutants, nuclear integration and to determinewhether the integration of foreign genes had occurred in the chloroplastgenome at the directed site by homologous recombination. The strategy todistinguish between nuclear and chloroplast transgenic plants was toland one primer (3P) on the native chloroplast genome adjacent to thepoint of integration and the second primer (3M) on the aadA gene (FIG.1). This primer set generated 1.6 kb PCR product in chloroplasttransformants (FIG. 4). Because this product cannot be obtained innuclear transgenic plants, the possibility of nuclear integration can beeliminated. PCR screening for chloroplast transformants after the firstculture cycle showed that 11 out of 15 betaine aldehyde resistant clonesintegrated foreign genes into the chloroplast genome. The rest of theresistant shoots may be either escapes or nuclear transformants. Hence,only PCR positive clones were advanced to further steps of regeneration.In contrast, nearly 60% of the spectinomycin resistant clones weremutants. Other labs have recently reported as high as 90% mutants amongspectinomycin resistant clones (Eibl et al. 1999; Sidorov et al. 1999).

Southern blot analysis was performed using total DNA isolated fromtransgenic and wild type tobacco leaves. Total DNA was digested with asuitable restriction enzyme. Presence of a BgllI cut site at the 3′ endof the flanking 16S rRNA gene and the trnA intron allowed excision ofpredicted size fragments in the chloroplast transformants anduntransformed plants. To confirm foreign gene integration andhomoplasmy, individual blots were probed with the flanking chloroplastDNA sequence (probe 1, FIG. 5A). In the case of the BADH integratedplastid transformants, the border sequence hybridized with a 7.29 kbpfragment while it hybridized with a native 4.47 kbp fragment in theuntransformed plants (FIG. 5B). The copy number of the integrated BADHgene was also determined by establishing homoplasmy in transgenic plants(Daniell et al. 1998; Guda et al. 2000). Tobacco chloroplasts containabout 10,000 copies of chloroplast genomes per cell. If only a fractionof the genomes was transformed, the copy number should be less than10,000. By confirming that the BADH integrated genome is the only onepresent in transgenic plants, it could be established that the BADH genecopy number could be as many as 10,000 per cell.

DNA gel blots were also probed with the BADH gene coding sequence (P2)to confirm specific integration into the chloroplast genomes andeliminate transgenic plants that had foreign genes also integrated intothe nuclear genome. In the case of the BADH integrated plants, the BADHcoding sequence hybridized with a 7.29 kbp fragment which alsohybridized with the border sequence in plastid transformant lines (FIG.5B). This shows that the BADH gene was integrated only into thechloroplast genome and not the nuclear genome in transgenic linesexamined in this blot. Also, this confirms that the tobaccotransformants indeed integrated the intact gene expression cassette intothe chloroplast genome and that no internal deletions or loop outsduring integration occurred via homologous recombination.

Osmoprotection.

In higher plants accumulation of osmoprotectants during salinity anddrought stress is a common phenomenon in their metabolic adaptation.Osmoprotectants help to protect plant organelles from osmotic shock aswell as the cellular membranes from damage during stress (Nuccio et al.1999). Among the osmoprotectants, glycine betaine is the most effectiveand is commonly present in a few families, including Chenopodiaceae andPoaceae. But most of the crop species including tobacco do notaccumulate glycine betaine. Since synthesis and localization of glycinebetaine is compartmentalized in chloroplasts, engineering thechloroplast genome for glycine betaine synthesis may provide an addedadvantage for chloroplast transgenic plants. BADH converts toxic betainealdehyde to non-toxic glycine betaine which is the second step in theformation of glycine betaine from choline. By analyzing BADH enzymeactivity, the expression of introduced BADH gene can be monitored. SinceBADH is a NAD+ dependent, enzyme activity is analyzed for the formationNADH. The reaction rate is measured by an increase in absorbency at 340nm resulting from the reduction of NAD+.

BADH enzyme activity was assayed in crude leaf extracts of wild type andtransgenic plants. Unlike previous reports, no purification withammonium sulfate was necessary in order to perform the BADH assay. Crudeextracts from chloroplast transgenic plants showed elevated activity(15-18 fold) compared to the untransformed tobacco (FIG. 6). The wildtype tobacco showed low endogenous activity as reported previously(Rathinasababathy et al. 1994). BADH enzyme activity was investigatedfrom young (top 3-4 leaves), mature (large well developed), developingleaves (in between young and mature) and bleached old leaves fromtransgenic plants. Crude leaf extracts from different developmentalstages of the same transgenic plant showed differential activity withthe most activity observed in mature leaves (18 fold over control) andleast activity in older leaves (15 fold over control, as seen in FIG.6). Unlike nuclear transgenic lines, crude extracts from differentchloroplast transgenic lines did not show significant variation in BADHactivity (data not shown).

Lack of pleiotropic effects. Expression of BADH and resultantaccumulation of glycine betaine did not result in any pleiotropiceffects; transgenic plants are morphologically indistinguishable fromcontrol untransformed plants (FIG. 7). They grew normally, flowered andset seeds. Germination of seeds from untransformed plants in thepresence of spectinomycin resulted in complete bleaching whereas seedsfrom the chloroplast transgenic plants germinated and grew normally(FIG. 8). Because untransformed seeds germinated in very highconcentrations of betaine aldehyde (10-15 mM), no comparison betweencontrol and transgenic seeds could be made during germination on betainealdehyde. This may be due to the presence of an active endogenous BADHor similar enzymatic activity in non-green plastids during germination.These results demonstrate that the introduced trait is stably inheritedin the subsequent generation and that it is safe to use betaine aldehydeselection because of the lack of pleiotropic effects.

Application to Other Plants. This invention applies to any higherplants, such as monocotyledonous and dicotyledonous plant species. Theplants that may be transformed via the universal vector with aantibiotic-free selectable marker maybe solanacious plants or plantsthat grow underground. Most importantly, this invention is applicable tothe major economically important crops such as maize, rice, soybean,wheat, and cotton. A non-exclusive list of examples of higher plantswhich may be so transformed include cereals such as barley, corn, oat,rice, and wheat; melons such as cucumber, muskmelon, and watermelon;legumes such as bean, cowpea, pea, peanut; oil crops such as canola andsoybean; solanaceous plants such as tobacco; tuber crops such as potatoand sweet potato; and vegetables like tomato, pepper and radish; fruitssuch as pear, grape, peach, plum, banana, apple and strawberry; fibercrops like the Gossypium genus such as cotton, flax and hemp; and otherplants such as beet, cotton, coffee, radish, commercial flowing plants,such as carnation and roses; grasses, such as sugar cane or turfgrass;evergreen trees such as fir, spruce, and pine, and deciduous trees, suchas maple and oak.

The invention is exemplified in the following non-limiting examples.

EXAMPLE 1

A. Betaine Aldehyde Selection of Tobacco Chloroplast Transformation.Tobacco plant chloroplasts were transformed by the universal vectorcontaining both a targeted gene of interest and the spinach betainealdehyde dehydrogenase gene. The transformed cells were cultured ingrowth medium containing betaine aldehyde, a phytotoxic aldehyde. Sincebetaine aldehyde is lethal to all untransformed cells, such cells willnot be regenerated in the growth medium. Therefore, all cells which growin the growth medium containing betaine aldehyde are successfullytransformed with the BADH gene lore importantly, such cells aresuccessfully transformed with the targeted gene of interest.

B. Other possible plants. Other than tobacco, this invention can bepracticed upon other monocotyledonous and dicotyledonous plants,including maize, rice, soybean, wheat, cotton, oat, barley, cucumber,muskmelon, watermelon, bean, cowpea, pea, peanut, canola, potato andsweet potato; tomato, pepper, radish, pear, grape, peach, plum, banana,apple, strawberry, flax, hemp, beet, coffee, radish, commercial flowingplants, such as carnation and roses; grasses, such as sugar cane orturfgrass; fir, spruce, and pine, maple and oak.

C. Other antibiotics that can be replaced. This example provides thatthe invention can replace all antibiotics as a selectable marker,including those listed in Molecular Biotechnology by Glick andPasternak, page 437, Table 17.4.

D. Other targeted genes of interest. This invention provides that genesof interest expressing desirable traits are encoded by the targeted DNAsequence in the expression cassette.

EXAMPLE 2

Other antibiotic-free phytotoxic agents include phytotoxic aldehydessuch as acetaldehyde, formaldehyde, proprionaldehyde, and butyraldehyde;herbicides such as triazines and cyanamide, including those listed inMolecular Biotechnology by Glick and Pasternak, page 459, Table 18.4.Also useful is light selection.

EXAMPLE 3

Other Genes of interest may be isolated from other organisms such asSugar Beet and E. Coli.

EXAMPLE 4

Other Promoters can be used to drive expression of the genes, includingthe psbA promoter, the accD promoter, the 16SrRNA promoter, and thoselisted in U.S. Pat. No. 5,693,507 and International Publication No.WO99/10513, both to Daniell.

EXAMPLE 5

Other chloroplast vectors may be used in lieu of the universal vector,including those listed in U.S. Pat. Nos. 5,693,507 and 5,932,479 toDaniell.

EXAMPLE 6

Targeted Genes of Interest include: Polypepide pro-insulin, PBPsynthetic polymer, Insulin, Human Serum Albumin, and Herbicideglyphosate. Other genes of interest include, but are not limited to theaminoglycosides listed in “Aminoglycosides: A Practical Review” byGonzalez, L. S. and Spencer, J. P., American Family Physician, No. 8,58:1811.

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1. A method of selecting for a transformed plant cell of a species thatis naturally inhibited by betaine aldehyde, wherein said method does notrequire selection for successful transformants by detection ofantibiotic resistance, said method comprising introducing into a plastidgenome of a starting plant cell of said species an integration andexpression cassette which comprises, as operably linked components, a 5′end of a plastid DNA spacer sequence, a 5′ untranslated region (UTR)having a ribosome binding site, a DNA sequence encoding a plant betainealdehyde dehydrogenase capable of detoxifying a betaine aldehyde in saidtransformed plant cell, a 3′ regulatory region functional in saidplastid, and a 3′ end of a plastid DNA spacer sequence, wherein saidmethod further comprises culturing said plant cell in a plant growthmedium comprising said betaine aldehyde, and selecting said transformedplant cell comprising said plastid genome in which the DNA sequenceencoding a detoxifying enzyme is expressed and hence is capable ofgrowth in the presence of said betaine aldehyde.
 2. The method of claim1, further comprising introducing into said plastid genome at least oneheterologous DNA sequence coding for a molecule of interest, said atleast one heterologous DNA sequence being located between the 5′ end and3′ end of the plastid DNA spacer sequence.
 3. The method of claim 1,wherein said method further comprises regenerating a transformed plantfrom said transformed plant cell.
 4. The method of claim 1, wherein saidDNA sequence encoding a detoxifying enzyme is from sugar beet or spinachplants.
 5. The method of claim 1, said 5′ UTR comprising a promoter froma gene selected from the group consisting of 16S rRNA, psbA, accD andatpB.
 6. The method of claim 1, wherein said plant betaine alddehydedehydrogenase is a spinach betaine aldehyde dehydrogenase.
 7. The methodof claim 1, wherein said plant species is tobacco.
 8. A method ofselecting for a transformed plant cell, said method comprisingintroducing into a plastid genome of a plant cell a transgene encoding aplant enzyme capable of detoxifying a non-antibiotic phytotoxic agent,wherein said method further comprises culturing said plant cell in aplant growth medium containing said non-antibiotic phytotoxic agent, andselecting said transformed plant cell that is capable of growth in thepresence of said phytotoxic agent due to expression of said transgeneinside a plastid, wherein said phytotoxic agent is a lethal aldehyde. 9.The method of claim 8, further comprising introducing into said plastidgenome at least one heterologous polyncleotide, wherein saidpolynucleotide encodes a molecule of interest.
 10. The method of claim8, wherein said method further comprises regenerating a transformedplant from said transformed plant cell.